4. Conclusions
In this paper, we have described the detection of a small molecule, the adenosine, from a sandwich assay with split-aptamers based on a biosensor/microarray with SPRi detection. The combination of signal amplification and sequence engineering allowed us to obtained state-of-the-art performances for small molecules detection by SPR (LOD = 50 nM). Those results have been possible due to an efficient grafting strategy of both gold surfaces (SPRi biosensor prisms and gold nanoparticles). The grafting of long PEG molecules along with the oligonucleotide probes reduces the non-specific adsorption and significantly decreased the background noise. Furthermore, the use of gold nanoparticles grafted with a common Split-APT sequence allowed us not only to amplify the SPRi signal but also to test various aptamer probe sequences on the microarrays (APT4, APT8, Split-APT4 and Split-APT8). The Split-APT4 sequence gave better results than Split-APT8 (three orders of magnitude difference in LOD) whereas APT4 and APT8 sequences were not suitable to detect adenosine in sandwich assay with Split-APT grafted AuNPs. Although some cooperative effects may occur due to a large number of Split-APT sequences present on each AuNPs, the sequence engineering for the Split-APT4 and Split-APT8 enables to avoid background signal in absence of adenosine for the former sequence. The good performance of Split-APT4 probe is explained in part by the signal OFF—signal ON behavior of the biosensor. 
Furthermore, it is interesting to notice that the LOD obtained with this sandwich strategy is also two orders of magnitude lower than the dissociation constant of the binding of adenosine with the complete aptamer APT4 (KD = 6 μM) [33,34]. AuNPs amplification of SPRi signal alone may not explain this impressive result. In fact, the size of the AuNP, 20 nm, and the number of Split-APT oligonucleotides per nanoparticles (few thousands) may lead to multiple interactions between Split-APT sequences on the same AuNP and Split-APT4 probes on the biosensor surfaces. The improved LOD is then related to the resulting cooperative behavior.
In conclusion, we have proven the interest of Surface Plasmon Resonance imaging for small molecules detection in a sandwich assay with split-aptamers. The microarrays format should allow us to generalize this approach to the multiple detection of small molecules in parallel.